The Function of FGFR1 Signalling in the Spinal Cord: Therapeutic Approaches Using FGFR1 Ligands after Spinal Cord Injury.

Extensive research is ongoing that concentrates on finding therapies to enhance CNS regeneration after spinal cord injury (SCI) and to cure paralysis. This review sheds light on the role of the FGFR pathway in the injured spinal cord and discusses various therapies that use FGFR activating ligands to promote regeneration after SCI. We discuss studies that use peripheral nerve grafts or Schwann cell grafts in combination with FGF1 or FGF2 supplementation. Most of these studies show evidence that these therapies successfully enhance axon regeneration into the graft. Further they provide evidence for partial recovery of sensory function shown by electrophysiology and motor activity evidenced by behavioural data. We also present one study that indicates that combination with additional, synergistic factors might further drive the system towards functional regeneration. In essence, this review summarises the potential of nerve and cell grafts combined with FGF1/2 supplementation to improve outcome even after severe spinal cord injury.

Myelinated and unmyelinated nerve fibers reinnervate tissue-engineered dermo-epidermal human skin analogs in an in vivo model.

PURPOSE: The clinical application of autologous tissue-engineered skin analogs is an important strategy to cover large skin defects. Investigating biological dynamics, such as reinnervation after transplantation, is essential to improve the quality of such skin analogs. Previously, we have examined that our skin substitutes are reinnervated by host peripheral nerve fibers as early as 8 weeks after transplantation. Here, we wanted to investigate the presence and possible differences regarding myelinated and unmyelinated host nerve fibers 15 weeks after the transplantation of light and dark human tissue-engineered skin analogs. METHODS: Human epidermal keratinocytes, melanocytes, and dermal fibroblasts were isolated from human light and dark skin biopsies. Keratinocytes and melanocytes were seeded on fibroblast-containing collagen type I hydrogels after expansion in culture. After additional culturing, the tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds created on the back of immuno-incompetent rats. Skin substitutes were excised and analyzed 15 weeks after transplantation. Histological sections were examined with regard to the ingrowth pattern of myelinated and unmyelinated nerve fibers into the skin analogs using markers, such as Substance P, NF200, and S100-Beta. RESULTS: We found myelinated and unmyelinated peripheral host nerve fibers 15 weeks after transplantation in the dermal part of our human skin substitutes. In particular, we identified large-diameter-myelinated Aß- and Aδ-fibers, and small-diameter C-fibers. Furthermore, we observed myelinated nerves in close proximity to CD31-positive blood capillaries. In the long run, both types of ingrown host fibers showed an identical pattern in both light and dark skin analogs. CONCLUSION: Our data suggest that myelinated and unmyelinated peripheral nerves reinnervate human skin substitutes in a long-term in vivo transplantation assay. Our tissue-engineered skin analogs attract A- and C-fibers to supply both light and dark skin analogs. Potentially, this process restores skin sensitivity and has, therefore, a significant relevance with regard to future application of autologous pigmented dermo-epidermal skin substitutes onto patients.

Type grouping in skeletal muscles after experimental reinnervation: another explanation.

Type grouping signifies clustering of muscle fibres of the same metabolic type, and is a frequent finding in reinnervated muscles. To elucidate the mechanism behind it, the rat sciatic nerve was either autografted or grafted with hollow synthetic nerve grafts. Twelve weeks later the number and fibre area of the type I and type II muscle fibres in the gastrocnemic and anterior tibial muscles were determined after ATP-ase staining. The number and diameter of peroneal nerve fibres distal to the grafts were measured, and the number of Aalpha-nerve fibres was derived. Nearly all nerve and muscle morphometrical parameters changed equally in both experimental groups. However, type grouping occurred frequently only after autografting, whereas the number of nerve fibres and the number of Aalpha-nerve fibres increased in this group. Hence type grouping cannot be explained by increased intramuscular sprouting subsequent to a decrease in the number of innervating nerve fibres, as previously presumed. Regenerating axons branch along their course through the peripheral nerve. We propose that the probability of the occurrence of type grouping is related to the dispersion of sibling branches in the nerve. In the autograft, emerging branches are kept together by Schwann cell basal lamina scaffolds, in contrast to the hollow synthetic nerve grafts where the emerging branches become dispersed. Thus, in muscles reinnervated after autografting, the probability that nerve branches that arrive at a specific muscle territory are sibling branches is greater than after hollow tube grafting. Consequently, the probability that type grouping will occur is greater.

Migrating and myelinating potential of neural precursors engineered to overexpress PSA-NCAM.

Polysialic acid (PSA) on NCAM is an important modulator of cell-cell interactions during development and regeneration. Here we investigated whether PSA overexpression influences neural cell migration and myelination. We stably expressed a GFP-tagged polysialytransferase, PSTGFP, in mouse neurospheres and induced prolonged PSA synthesis. Using a chick xenograft assay for migration, we show that PSA can instruct precursor migration along the ventral pathway. PSA persistence did not change neural precursor multipotentiality in vitro but induced a delay in oligodendrocyte differentiation. PSTGFP+ precursors showed widespread engraftment in shiverer brain, closely similar to that observed with control precursors expressing a fluorescent protein. Initially, myelination by oligodendrocytes was delayed but, eventually, down-regulation of PSTGFP occurred, allowing myelination to proceed. Thus down-regulation of polysialyltransferases takes place even in cells where its RNA is under the control of a heterologous promoter and engineering PSA overexpression in neural precursors does not cause irreversible unphysiological effects.

Effect of number of fascicle on axonal regeneration in cable grafts.

The effect of number of fascicles on axonal regeneration in cable grafts was examined in the rat cable graft model. The study comprised three experimental groups: the 5f-group, which received 5 fascicles, larger than the host; the 3f-group, in which the total area of the graft fascicles was similar to that of the host; and the 1f-group, which received one fascicle cable graft, smaller in diameter than the host nerve. At the graft segment, well-myelinated fibers were observed both inside and outside the graft fascicles. The three groups showed no difference in morphometric and functional assessment, suggesting that the fibers which regenerated through the outside of the graft might be effectively induced into the distal host. The disproportionate enlargement of the graft fascicle of the 1f-group also increased the fibers passing through it. These findings suggest that a small number of fascicles can induce a larger population of regenerated fibers in the 20-mm cable graft model.

The use of a muscle graft to repair a segmentary nerve defect. An experimental study using the sciatic nerve of rats as model.

The use of a devitalized skeletal muscle graft and conventional nerve graft to repair a 5mm long segmentary sciatic nerve lesion was studied in rats by means of functional, morphometric and spinal cord motor neuron cell response evaluation. Thirty-four rats were used and divided into four groups according to the procedure: (1) sham operation; (2) conventional nerve grafting; (3) muscle grafting; (4) unrepaired lesion. The sciatic functional index (SFI) was evaluated every fortnight up to the 105th postoperative day by measuring three parameters in the rats' footprint. The animals of Groups 2 and 3 presented initial complete functional loss, followed by slow but steady recovery, with final similar SFIs. The histologic and morphometric studies showed an increased small diameter/thin myelin sheath nerve fiber density distally to the lesion site for both types of graft. An increased population of motor neurons was observed in the anterior horn of the lumbar spinal cord segment with both types of grafts, but not in the control groups. The SFI, histologic and morphometric data did not differ significantly between the two types of graft, thus indicating a similar behavior. The authors conclude that a 5mm long skeletal muscle graft works as well as a conventional nerve graft.

CNS axons retain their competence for myelination throughout life.

An important question relevant to developing remyelination therapies is whether axons that remain without myelin sheaths after an episode of demyelination retain myelination competence. To resolve this, we have developed a model of transplantation into the nerve fibre layer of the adult rat retina, where the axons are unmyelinated. In the adult, these axons can be myelinated by transplantation of both the oligodendrocyte progenitor cells (OPCs) and an OPC line (CG4). The extent of myelination achieved following transplantation of OPCs is the same in young adult recipients (2 months old) as that which occurs in old adult recipients (12-18 months old), indicating that there are no changes in axons remaining unmyelinated for many months that would prevent effective remyelination. This finding suggests that chronically demyelinated regions of axons such as those in seen in multiple sclerosis are likely to remain competent to be remyelinated.

Comparative analysis of remyelinating potential of focal and intravenous administration of autologous bone marrow cells into the rat demyelinated spinal cord.

The remyelinating potential of autologous bone marrow cells was studied after direct injection and following intravenous injection into rats with a demyelinated lesion in the spinal cord. Both focal and intravenous injections of acutely isolated mononuclear bone marrow cell fractions resulted in varying degrees of remyelination. Suspensions of bone marrow cells collected from the same rat were delivered at varied concentrations (10(2) to 10(5) for direct injection and 10(4) to 10(7) for i.v. injections). The lesions were examined histologically 3 weeks after transplantation. Light microscopic examination revealed remyelination in the dorsal funiculus with both injection protocols, but the extent of remyelination was proportional to the number of injected cells. To attain the same relative density of remyelination achieved by direct injection, intravenous administration of cells required delivery of substantially more cells (two orders of magnitude). However, the availability of autologous bone marrow cells in large number and the potential for systemically delivering cells to target lesion areas without neurosurgical intervention suggest the potential utility of intravenous cell delivery as a prospective therapeutic approach in demyelinating disease.

Comparative dose-dependence study of FK506 on transected mouse sciatic nerve repaired by allograft or xenograft.

We evaluated the effects of FK506, at doses of 0.2, 2, and 5 mg/kg/day, on the response to nerve grafts implanted in outbred mice. A 6 mm long segment of the sciatic nerve was transected and repaired by autograft (the same segment resected), allograft (from another mouse), or xenograft (from a rat nerve). The regenerating nerves were harvested after 3 weeks and studied under light and electron microscope. Allografts of animals treated with the 5 mg/kg/day dose of FK506 appeared similar to those from autografts, demonstrating an equivalent number of myelinated fibers. In mice treated with the 2 mg/kg/day dose, regeneration was slightly hindered, as indicated by the reduced number of myelinated fibers. In contrast, in mice given a 0.2 mg/kg/day dose of FK506, allografts were not different from untreated allografts; both groups showed a marked rejection response with only few unmyelinated axons and no myelinated fibers. Xenografts showed a more severe rejection than allografts, with a marked inflammatory cell reaction throughout the graft. In contrast, in mice treated with the 5 mg/kg/day dose, xenografts exhibited a mild cell reaction and a greater number of regenerated myelinated fibers. In conclusion, effective axonal regeneration is achieved with FK506 administration at doses of 5 mg/kg/day through allografts and, partially, through xenografts.

Evidence for impaired axonal regeneration in PMP22 duplication: studies in nerve xenografts.

Whether axonal regeneration in Charcot-Marie-Tooth (CMT) neuropathies is impaired has not been addressed in detail. Our studies in nude mice harboring xenografts from patients with different primary Schwann cell (SC) genetic defects suggested an intimate association between the onset of myelination and impairment in the growth capacity of nude mice axons engulfed by the mutant SCs. To assess the effects of peripheral myelin protein 22 (PMP22) gene duplication on the regeneration process, we conducted morphometric studies to generate temporal growth profiles of myelinated axons within the xenografts obtained from CMT1A patients and from healthy controls. Axon size distribution histograms in controls at different time intervals revealed that size differentiation of myelinated fibers within the grafts is established as early as 2 weeks, and that the temporal pattern of myelination of different sized axons has striking similarities to myelination during development. In PMP22 duplication grafts, the onset of myelination is delayed and the regeneration capacity of all fiber sizes is impaired. This defect, however, is most pronounced for the large diameter axons. In addition, significant large fiber loss occurred after 12 weeks with a concomitant new cycle of regeneration of small size axons. These studies show that the PMP22 duplication in SCs have profound effects on the regeneration process, which might be a contributing factor to preferential distal axonal loss.

Grafted swine neuroepithelial stem cells can form myelinated axons and both efferent and afferent synapses with xenogeneic rat neurons.

Neuroepithelial stem cells derived from the swine mesencephalic neural tube were examined regarding their eligibility for neural xenografting as a donor material, with the aim of evaluating myelinated axon formation and both types of synaptic formation with xenogeneic host neurons as part of possible neural circuit reconstruction. The mesencephalic neural tube tissues were dissected out from swine embryos at embryonic days 17 and 18 and were implanted immediately into the striatum of the Parkinsonian model rat. The swine-derived grafts had many nestin-positive rosette-forming, neurofilament-positive, and tyrosine hydroxylase-positive cells in the rat striatum. Electron microscopic study revealed both efferent and afferent synaptic formations in the donor-derived immature neurons or tyrosine hydroxylase-positive donor cells in the grafts. Myelinated axons, both positive and negative for swine-specific neurofilament antibody, were mingled together in the graft. These results indicated that implanted neuroepithelial stem cells could survive well and divide asymmetrically into both nestin-expressing precursors and differentiated neurochemical marker-expressing neurons in the xenogeneic rat striatum, with the help of an immunosuppressant. Donor-derived immature neurons formed both efferent and afferent synapses with xenogeneic host neurons, and donor-derived axons were myelinated, which suggests that implanted swine neuroepithelial stem cells could possibly restore damaged neuronal circuitry in the diseased brain.

Estudo sobre a influência da sutura distal em enxertos longos de nervo com emprego de técnica microcirúrgica / Study on the influence of distal suture in long nerve grafts using a microsurgery technique

Foram operados 14 coelhos nos quais se realizaram enxertos longos de nervo fibular do tipo autólogo, usando-se técnica microcirúrgica. Num grupo (sete coelhos), as suturas proximal e distal foram feitas no mesmo estágio cirúrgico e, no outro (sete coelhos), a sutura distal foi realizada 60 dias após a sutura proximal. A análise dos resultados näo demonstrou haver diferença estatisticamente significante no número de fibras mielinizadas no segmento proximal, no enxerto propriamente dito e no segmento distal em ambos os grupos

The dorsal approach in harvesting the second toe.

The dorsal approach, with early section of the second metatarsal bone, allows direct access to the plantar structures of the second toe (arteries, nerves, and flexor tendons). A "multiple set arterial transfer" then becomes possible, improving the blood supply to the toe. Early walking is encouraged on the fifth postoperative day when the patient leaves the hospital. No vascular complications have occurred in any microvascular reconstruction after the fifth postoperative day. Close approximation of the first and third toe of the donor foot not only improves the overall appearance of the foot, but also avoids a possible hallux valgus deformity as a postoperative complication.

Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and radioautography.

Cross-anastomoses and autogenous grafts of unmyelinated and myelinated nerves were examined by electron microscopy and radioautography to determine if Schwann cells are multipotential with regard to their capacity to produce myelin or to assume the configuration seen in unmyelinated fibres. Two groups of adult white mice were studied. (A) In one group, the myelinated phrenic nerve and the unmyelinated cervical sympathetic trunk (CST) were cross-anastomosed in the neck. From 2 to 6 months after anastomosis, previously unmyelinated distal stumps contained many myelinated fibres while phrenic nerves joined to proximal CSTs became largely unmyelinated. Radioautography of distal stumps indicated that proliferation of Schwann cells occurred mainly in the first few days after anastomosis but was also present to a similar extent in isolated stumps. (B) In other mice, CSTs were grafted to the myelinated sural nerves in the leg. One month later, the unmyelinated CSTs became myelinated and there was no radioautographic indication of Schwann cell migration from the sural nerve stump to the CST grafts. Thus, Schwann cell proliferation in distal stumps is an early local response independent of axonal influence. At later stages, axons from the proximal stumps cause indigenous Schwann cells in distal stumps from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously myelinated nerves become associated with unmyelinated fibres. Consequently, the regenerated distal nerve resembled the proximal stump. It is suggested that this change is possible because Schwann cells which divide after nerve injury reacquire the developmental multipotentiality which permits them to respond to aoxonal influences.

Development of a receptor on a foreign nerve fiber in a Pacinian corpuscle.

When the sensory fiber of a Pacinian corpuscle (in cat mesentery) is transected (at the inferior mesenteric nerve) transduction fails within 30 hours: the nerve ending produces no generator potentials in response to mechanical stimulation. Electrically elicited nerve impulse conduction continues for at least another 18 hours. A transducer mechanism develops on a regenerating nerve fiber when this fiber enters the denervated corpuscle. Such transducer development takes place on myelinated fibers from the inferior mesenteric nerve, which normally supplies corpuscles, as well as on myelinated hypogastric nerve fibers, which normally do not go to corpuscles, including fibers larger than the original corpuscle afferents.